flavin-adenine-dinucleotide and benzeneboronic-acid

A method is provided for the recognition of glycated molecules based on their binding affinities to boronate-carrying monolayers. The affinity interaction of flavin adenine dinucleotide (FAD) and horseradish peroxidase (HRP) with phenylboronic acid monolayers on gold was investigated by using voltammetric and microgravimetric methods. Conjugates of 3-aminophenylboronic acid and 3,3'-dithiodipropionic acid di(N-hydroxysuccinimide ester) or 11-mercaptoundecanoic acid were prepared and self-assembled on gold surfaces to generate monolayers. FAD is bound to this modified surface and recognized by a pair of redox peaks with a formal potential of -0.433 V in a 0.1 M phosphate buffer solution, pH 6.5. Upon addition of a sugar to the buffer, the bound FAD could be replaced, indicating that the binding is reversible. Voltammetric, mass measurements, and photometric activity assays show that the HRP can also be bound to the interface. This binding is reversible, and HRP can be replaced by sorbitol or removed in acidic solution. The effects of pH, incubation time, and concentration of H(2)O(2) were studied by comparing the catalytic reduction of H(2)O(2) in the presence of the electron-donor thionine. The catalytic current of the HRP-loaded electrode was proportional to HRP concentrations in the incubation solution in the range between 5 microg mL(-1) and 0.1 mg mL(-1) with a linear slope of 3.34 microA mL mg(-1) and a correlation coefficient of 0.9945.

Topics: Boronic Acids; Flavin-Adenine Dinucleotide; Horseradish Peroxidase; Molecular Structure

The preparation of integrated, electrically contacted, flavoenzyme and NAD(P)(+)-dependent enzyme-electrodes is described. The reconstitution of apo-glucose oxidase, apo-GOx, on a FAD cofactor linked to a pyrroloquinoline quinone (PQQ) phenylboronic acid monolayer yields an electrically contacted enzyme monolayer (surface coverage 2.1 x 10(-)(12) mol cm(-)(2)) exhibiting a turnover rate of 700 s(-)(1) (at 22 +/- 2 degrees C). The system is characterized by microgravimetric quartz-crystal microbalance analyses, Faradaic impedance spectroscopy, rotating disk electrode experiments, and cyclic voltammetry. The performance of the enzyme-electrode for glucose sensing is described. Similarly, the electrically contacted enzyme-electrodes of NAD(P)(+)-dependent enzymes malate dehydrogenase, MalD, and lactate dehydrogenase, LDH, are prepared by the cross-linking of affinity complexes generated between the enzymes and the NADP(+) and NAD(+) cofactors linked to a pyrroloquinoline quinone phenylboronic acid monolayer, respectively. The MalD enzyme-electrode (surface coverage 1.2 x 10(-)(12) mol cm(-)(2)) exhibits a turnover rate of 190 s(-)(1), whereas the LDH enzyme-electrode (surface coverage 7.0 x 10(-)(12) mol cm(-)(2)) reveals a turnover rate of 2.5 s(-)(1). Chronoamperometric experiments reveal that the NAD(+) cofactor is linked to the PQQ-phenylboronic acid by two different binding modes. The integration of the LDH with the two NAD(+) cofactor configurations yields enzyme assemblies differing by 1 order of magnitude in their bioelectrocatalytic activities.

Topics: Animals; Aspergillus niger; Boronic Acids; Chickens; Electric Impedance; Electrochemistry; Electrodes; Flavin-Adenine Dinucleotide; Glucose Oxidase; Gold; Kinetics; L-Lactate Dehydrogenase; Liver; Malate Dehydrogenase; NADP; Rabbits